Physiologically active substance PF1191 and process for producing the same

ABSTRACT

A substance PF1191 having an inhibitory activity to kainic acid toxicity represented by the following formula (I) which is obtained by incubating a fungus belonging to the genus Eupenicillium and isolating the product thus produced from the culture by solvent extraction, adsorption column chromatography, gel filtration, etc.

This application is a 371 of PCT/JP98/01046 filed Mar. 12, 1998.

TECHNICAL FIELD

This invention relates to a novel physiologically active substancePF1191 showing an inhibitory activity to kainic acid toxicity and thusbeing useful as a remedy and a preventive for diseases caused by nervousdisturbances or its salt, and a process for producing the same.

BACKGROUND ART

Excitatory amino acids (glutamic acid, aspartic acid, etc.) playimportant roles as neurotransmitters in mammals. On the other hand,there have been reported findings one after another that abnormalexcitation of excitatory amino acid receptors is one of the causativefactors of nerve cell death occurring in brain ischemia, head injury,Alzheimer's disease, Parkinson's disease, Huntington's chorea, etc. Itis also suggested that abnormalities in these excitatory amino acidreceptors participate in the onset of schizophrenia. Under thesecircumstances, studies have been vigorously made on excitatory aminoacid receptor antagonists and agonists.

Regarding substances with antagonism to excitatory amino acid receptorswith microbial origin, it is reported that a substance ES-242 isproduced by Verticillium sp. ES-242 (J. Antibiotics, vol. 45, 88-93(1992)).

To develop an antagonist to excitatory amino acid receptors, the presentinventors have studied and searched to find a substance originating in amicrobial product which is capable of inhibiting the toxicity of kainicacid on brain nerve cells.

It is also reported that kainic acid is toxic to primarily culturedchick telencephalon nerve cells and this toxicity by the excitatoryamino acid is expressed via the cystine transporter system andnon-N-methyl-D-aspartic acid (NMDA) receptor (Neuroscience Letters, vol.139, 205-208 (1992)). This toxicity is inhibited by non-NMDA receptorantagonists including 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX),6,7-dinitroquinoxaline-2,3-dione (DNQX) and6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX).

Accordingly, the present invention aims at providing a substance whichis capable of inhibiting the toxicity of kainic acid on brain nervecells and therefore useful as a remedy and a preventive for diseasescaused by nervous disturbances.

DISCLOSURE OF THE INVENTION

By using primarily cultured chick telencephalon nerve cells, the presentinventors searched microbial products to find out a substance capable ofinhibiting the toxicity of kainic acid on brain nerve cells. As aresult, they found that a strain belonging to the genus Eupenicilliumaccumulated a product having an inhibitory activity to kainic acidtoxicity in its culture medium. Next, the above product (i.e., thesubstance PF1191) was isolated and purified from the culture andidentified as a novel substance based on its physicochemical properties.As the results of the subsequent studies, it was confirmed that thissubstance PF1191 has a stronger inhibitory activity to kainic acidtoxicity than that of DNQX. The present invention has been completedbased on these findings.

Accordingly, the first gist of the present invention resides in theprovision of a novel substance PF1191, which is represented by thefollowing formula (I) and involves all stereoisomers thereof, or itssalt:

The second gist of the present invention resides in the provision of aprocess for producing the physiologically active substance PF1191 or itssalt which comprises incubating a fungus belonging to the genusEupenicillium and being capable of producing the substance PF1191, andcollecting the thus produced substance PF1191 from the culture.

MODE FOR CARRYING OUT THE INVENTION

The microorganism to be used in the production of the substance PF1191in the present invention may be an arbitrary one so long as it belongsto the genus Eupenicillium and is capable of producing the substancePF1191. As an example of the microorganism usable in the presentinvention, a strain Eupenicillium shearii PF1191 (hereinafter referredto as the “PF1191 strain”) having been newly isolated from the soil maybe cited.

1. Mycological Properties of PF1191 Strain

(1) Characteristics in Incubation

Colonies grow well on Czapek-yeast extract agar and attain 35 to 40 mmin diameter after incubating at 25° C. for 7 days. They are a white tograyish brown, velvety, radial wrinkled and consisting of a thickmycelial layer. A number of ascomata are formed in submerged hyphae. Ithas a back face in a pale olive color.

Colonies grow well on malt extract agar and attain 18 to 20 mm indiameter after incubating at 25° C. for 7 days. They are pale brown,velvety, flat and consisting of thin mycelial layer. A number ofascomata are formed in submerged hyphae. It has a back face in a paleorange color.

Colonies grow well on oatmeal agar and attains 18 to 20 mm in diameterafter incubating at 25° C. for 7 days. They are grayish brown, velvety,flat and consisting of thin mycelial layer. A number of ascomata areformed on the colony surface to give a granular appearance. It has aback face in a pale yellowish brown color.

At an incubation temperature of 37° C., the growth of the strain isinferior in each of the above-described media to the growth at 25° C.

(2) Morphological Characteristics

Cleistochecia are globose to ellipsoidal, yellowish brown and 150 to 350μm in diameter. Each peridium is composed of sclerenchyma cells andturns from pseudoparenchyma into sclerotium. They take 3 to 4 weeks formaturation. Asci are globose to ellipsoidal in shape, 6 to 8×5 to 6.5 μmin size, 8-spored and evanescent at maturity. Ascospores are lens-shapedand 3.0 to 3.5×2.0 to 3.0 μm in size, each having two equatorial crestsand fine projections on the convex surface. Conidiophores are 200 to600×2 to 2.5 μm in size and smooth-walled. Penicilli are biverticillate,metulae are 12 to 20×2.5 to 3 μm in size and 2 to 4 per stipe, andphialides are ampulliform, 7 to 12×2.5 to 3 μm in size and 5 to 7 permetula. Conidia are ellipsoidal in shape and 2.5 to 3×2 to 2.5 μm insize and smooth-wallod.

Based on these mycological characteristics, this strain has beenidentified as Eupenicillium shearii belonging to Plectomycetes and namedEupenicillium shearii PF1191 strain. In the identification, use was madeof “The ascomycete genus Eupenicillium and related Penicillium anamorphs(Studies in Mycology, No. 23)”, Amelia C. Stolk and Robert A. Samson,Centraal-bureau voor Schimmelcultures, 1983.

This PF1191 strain has been deposited as follows.

{circle around (1)} Depository name: International Depository, NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology

Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi, 305-8566 JAPAN

{circle around (2)} Deposition Date: Original deposition date: Dec. 2,1996 Request for transfer: Feb. 20, 1998 (transferred from FERM P-15973deposited on Dec. 2, 1996)

{circle around (3)} Deposition No. FERM BP-6263.

Similar to other funguses, the PF1191 strain is liable to undergochanges in its properties. Therefore, use can be made in the presentinvention of, for example, mutants (either spontaneous ones ormutagenized ones) originating in the PF1191 strain, conjugants or generecombinants, so long as they are capable of producing the substancePF1191.

2. Method for Incubating Substance PF1191-producing Strain

The microorganism capable of producing the substance PF1191 may beincubated in a medium containing nutrients commonly usable bymicroorganisms. As the nutrients, use can be made of thoseconventionally employed in the incubation of funguses. Examples ofcarbon sources usable herein include glucose, sucrose, starch syrup,dextrin, starch, glycerol, molasses, animal oils and vegetable oils.Examples of the nitrogen sources include organic matters such as soybeanmeal, wheat germ, corn steep liquor, cotton seed meal, meat extract,peptone, yeast extract, ammonium sulfate, sodium nitrate and urea.Furthermore, it is possible to add to the medium inorganic salts capableof providing ions of sodium, potassium, calcium, magnesium, cobalt,chlorine, phosphate, sulfate, etc. The medium may furthermore containappropriate organic and inorganic matters promoting the growth of thestrain and accelerating the production of the substance PF1191.

Regarding the incubation method, it is most desirable to employ aerobicculture, in particular, stand culture. Although the incubation may beappropriately performed at 25 to 30° C., it is carried out at around 26°C. in most cases. The productivity of the substance PF1191 variesdepending on the medium and incubation conditions. The accumulation ofthe substance PF1191 attains the maximum level usually after incubatingfor 2 to 14 days either in stand culture, shaking culture or tankculture. When the accumulation of the substance PF1191 in the liquidculture medium attains the maximum level, the incubation is ceased andthe target product is isolated and purified from the medium.

The substance PF1191 according to the present invention produced by theabove-described process is purified in the following manner.

By taking advantage of the properties, the substance PF1191 can beextracted and purified by using separating operations commonly employedin the art, for example, solvent extraction, ion exchange resin method,adsorption or partition column chromatography, gel filtration, dialysisand precipitation, either alone or appropriately combinedly. Moreparticularly speaking, a water-miscible solvent (methanol, ethanol,acetone, etc.) is added to the culture followed by stirring to give anextract of the substance PF1191. After evaporating off the organicsolvent from the extract, the target substance PF1191 with a high puritycan be purified from the residual aqueous solution by appropriatelycombining, for example, adsorption/desorption with the use of anadsorbent, molecular partition with the use of a gel filtration agent,recrystallization from an appropriate solvent, high-performance liquidchromatography, etc. For example, the substance PF1191 can be purifiedby subjecting the above-described aqueous solution successively toactivated charcoal column chromatography, gel filtration chromatography,and liquid chromatography with the use of an ODS column.

Salts of the substance PF1191 include metal salts (for example, alkalimetal salts such as sodium salt, alkaline metal salts such as calciumsalt, etc.) and base addition salts formed together withpharmaceutically acceptable inorganic or organic bases.

BEST MODE FOR CARRYING OUT THE INVENTION

Now, examples of the embodiment of the present invention will bedescribed. Since the present invention has clarified the properties ofthe substance PF1191, various processes for producing the substancePF1191 can be devised on the basis of its properties. Accordingly, itshould be understood that the present invention is not construed asbeing limited to these examples but involves not only modifications ofthe processes disclosed in the examples but also any methods forproducing, extracting and purifying the substance PF1191 with the use ofpublicly known operations based on the properties of the substancePF1191 having been disclosed by the present invention.

EXAMPLE 1

A medium (pH 7.0 before sterilization) consisting of 2.0% of starch,1.0% of glucose, 0.5% of peptone, 0.6% of wheat germ, 0.3% of yeastextract, 0.2% of soybean meal and 0.2% of calcium carbonate was used asa seed medium. A solid medium prepared by adding2.5% of soybean meal tosufficiently water-soaked rice was used as a production medium.

20 ml of the above-described seed medium was put into a 100 mlErlenmeyer flask followed by sterilization at 120° C. for 15 minutes.Next, one platinum loopful of the strain PF1191 grown on a slant agarmedium was inoculated thereinto and incubated under shaking at 25° C.for 4 days. Then 30 g of the above-described production medium was putinto another 100 ml Erlenmeyer flask followed by sterilization at 120°C. for 15 minutes. After inoculating with 1 ml of the seed culturedescribed above, the flask was well stirred and incubated as stationaryculture at 28° C. for 14 days. 3 kg of the thus obtained culture wasextracted with a 67% aqueous solution of acetone for 1 hour to give 10 Lof a cell extract.

EXAMPLE 2

After evaporating off acetone from the cell extract obtained as inExample 1, the active component contained in the residual aqueoussolution was adsorbed on an activated charcoal column (50 mm inradius×400 mm in height). After washing the activated charcoal columnwith water, the active component was eluted with a 50% aqueous solutionof acetone. The eluate thus obtained was concentrated and then adsorbedon a column (50 mm in radius×450 mm in height) packed with TOYOPEARLHW-40F (manufactured by Tosoh Corporation) and gel filtrationchromatography was performed by using a 60% aqueous solution of methanolas a developing solvent. After concentrating the active fractions, theactive component was adsorbed on a column (35 mm in radius×600 mm inheight) packed with PEGASIL ODS (manufactured by Senshu Science) andcolumn chromatography was performed with the use of a 20% aqueoussolution of methanol as a developing solvent. The active fractions thusobtained were combined and concentrated to give a crude powdercontaining the active component. This crude powder was further purifiedby high performance liquid chromatography (column: PEGASIL ODS, 20 mm inradius×250 mm in height) by using a 20 mM diethylamine carbonate buffersolution containing 5% of methanol as a developing solution. Thus, 9.2mg of the pure substance PF1191 was obtained.

The thus obtained substance PF1191 according to the present inventionhave the following properties.

(1) Color and form: Colorless powder.

(2) Molecular formula: C₁₈H₂₁N₃O₉Cl₂.

(3) Mass spectrum (HRFAB-MS): found 494.0757[M+H]⁺calcd. 494.0733.

(4) Melting point: 235-238° C. (decomp.).

(5) Specific rotation: [α]_(D) ²¹=−30.9° (c 0.7, H₂O).

(6) UV absorption spectrum:

In aq. solution λ_(max)(ε) 217(18,200), 297(8,200) nm.

In 0.01N HCl λ_(max)(ε) 214(23,200), 255(6,700) nm.

(7) Infrared absorption spectrum:

ν(KBr cm⁻¹): 3410, 1640, 1560, 1485, 1385.

(8) Nuclear magnetic resonance spectra: ¹H and ¹³C NMR spectra in D₂Oare as follows.

(a) ¹H NMR spectrum δ_(H)(ppm) 7.62(2H), 4.41(1H), 4.35(1H), 4.16(1H),3.70(1H), 2.41(1H), 2.28(1H), 2.12(1H), 2.06(1H), 2.01(1H), 1.61(1H).

(b) ¹³C NMR spectrum δ_(C)(ppm) 177.8 m 175.0, 171.3, 168.2, 153.8,128.6, 125.5, 123.0, 77.1, 71.4, 59.7, 56.2, 54.2, 35.6, 32.7, 30.4.

(9) High performance liquid chromatography:

Column: PEGASIL ODS (manufactured by Senshu Science, 4.6 mm indiameter×250 mm in height).

Mobile phase: 20 mM diethylamine carbonate buffer containing 5% ofmethanol.

Flow rate: 1 ml/min.

Detector: UV 300 nm.

Retention time: 6.1 min.

Based on these data, it is considered that the substance PF1191 has thefollowing structure.

TEST EXAMPLE

The inhibitory activities to kainic acid toxicity of the substancePF1191 on primarily cultured nerve cells of chick telencephalon and rathippocampus were examined. The results were as follows. The test withthe use of the primarily cultured nerve cells of chick telencephalon wascarried out in accordance with the method reported in NeuroscienceLetters, vol. 139, 205-208 (1982), while the test with the use of theprimarily cultured nerve cells of rat hippocampus was carried out inaccordance with the method reported in Brain Research, 126(1977)397-425.

The molar concentration at which the cytotoxicity caused by the additionof 500 μM of kainic acid to the cultured cells was inhibited at a ratioof 50% was expressed in EC₅₀ (effective concentration-fifty) in Table 1below.

TABLE 1 Inhibitory activity to kainic acid toxicity of the substancePF1191 50% Effective conc. (EC₅₀: μM) Cell and Agonist Substance PF1191DNQX primarily cultured nerve cells 1.5 7.5 of chick telencephalon +kainic acid (500 μM) primarily cultured nerve cells 0.45 1.8 of rathippocampus + kainic acid (500 μM) DNQX:6,7-dinitroquinoxaline-2,3-dione.

It was thus confirmed that the substance PF1191 showed strongerinhibitory activity to kainic acid toxicity in comparison with the knownsubstance DNQX in each test.

The novel substance PF1191 provided by the present invention has anactivity of inhibiting the toxicity of kainic acid on nerve cells and,therefore, is useful as a remedy and a preventive for diseases caused bynervous disturbances such as brain ischemia.

Industrial Applicability

The present invention provides a novel substance PF1191 having anactivity of inhibiting the toxicity of kainic acid on nerve cells. Thissubstance PF1191 is useful as a nerve cell protective agent againstnerve cytotoxicity of excitatory amino acids formed in, for example,brain ischemia.

What is claimed is:
 1. A substance PF1191 represented by the followingformula (I) or its salt:


2. A process for producing the substance PF1191 or its salt whichcomprises incubating a microorganism belonging to the genusEupenicillium and being capable of producing the physiologically activesubstance PF1191, and collecting the substance PF1191 as claimed inclaim 1 from the culture.